Selective deletion of Pten in theca-interstitial cells leads to androgen excess and ovarian dysfunction in mice.

Theca cell-selective Pten mutation (tPtenMT) in mice resulted in increases in PDK1 and Akt phosphorylation, indicating an over-activation of PI3K signaling in the ovaries. These mice displayed elevated androgen levels, ovary enlargement, antral follicle accumulation, early fertility loss and increased expression of Lhcgr and genes that are crucial to androgenesis. These abnormalities were partially reversed by treatments of PI3K or Akt inhibitor. LH actions in Pten deficient theca cells were potentiated. The phosphorylation of Foxo1 was increased, while the binding of Foxo1 to forkhead response elements in the Lhcgr promoter was reduced in tPtenMT theca cells, implying a mechanism by which PI3K/Akt-induced upregulation of Lhcgr in theca cells might be mediated by reducing the inhibitory effect of Foxo1 on the Lhcgr promoter. The phenotype of tPtenMT females is reminiscent of human PCOS and suggests that dysregulated PI3K cascade in theca cells may be involved in certain types of PCOS pathogenesis.

Mouse germline restriction of Oct4 expression by germ cell nuclear factor.

The POU-domain transcription factor Oct4 is essential for the maintenance of the mammalian germline. In this study, we show that the germ cell nuclear factor (GCNF), an orphan nuclear receptor, represses Oct4 gene activity by specifically binding within the proximal promoter. GCNF expression inversely correlates with Oct4 expression in differentiating embryonal cells. GCNF overexpression in embryonal cells represses Oct4 gene and transgene activities, and we establish a link to transcriptional corepressors mediating repression by GCNF. In GCNF-deficient mouse embryos, Oct4 expression is no longer restricted to the germ cell lineage after gastrulation. Our studies suggest that GCNF is critical in repressing Oct4 gene activity as pluripotent stem cells differentiate and in confining Oct4 expression to the germline.

Germ cell nuclear factor.

Germ cell nuclear factor (GCNF) is an orphan nuclear receptor for which a ligand has yet to be identified. However, we do know that GCNF binds to a novel response element as a homodimer and regulates expression of genes, such as the protamines, through this element. In the absence of a ligand, GCNF is a transcriptional repressor that interacts with co-repressors. During embryonic development, GCNF is expressed between the gastrula and neurula stages. Loss of GCNF causes embryonic lethality, disrupts normal somitogenesis, as well as neural tube and axis formation, suggesting that GCNF is a critical factor for normal embryonic development. In adult vertebrates, GCNF expression is predominantly found in the germ cells of gonads. GCNF expression in germ cells suggests that understanding its function in adults will yield greater insight into the regulation of gametogenesis, leading to new contraceptive targets.

Germ cell nuclear factor is a transcriptional repressor essential for embryonic development.

GCNF is an orphan member of the nuclear receptor superfamily. The nuclear receptor superfamily is a large superfamily of transcription factors, the majority of which are designated as orphan receptors because their ligands and functions are currently unknown. GCNF (Germ Cell Nuclear Factor) is so named because of its restricted expression pattern in the adult. In the testis, GCNF is expressed only in the post meiotic round spermatids. Likewise in the ovary, GCNF's expression is restricted to the growing oocyte. To date nothing is known of GCNF's putative ligand; however, much is known about its physiological function through the use of gene targeting. Inactivation of the GCNF gene showed that it was essential for normal embryonic development. In addition to being expressed in the germ cells of the adult, it is expressed widely throughout the embryo after gastrulation. Significant strides have also been made in understanding GCNF's mechanism of action using molecular biology. The DNA binding properties of GCNF have been investigated and its response element identified. GCNF binds as a homodimer to a direct repeat element with zero nucleotides between the reiterated sequence AGGTCA. GCNF target genes have been identified that contain this DR0 element in their promoters. Such genes as Protamines 1 and 2 and Oct4 are regulated by GCNF through this element. GCNF has been shown to be a repressor of the protamine and Oct4 genes. GCNF's repression function has been shown to be mediated by interaction with the co-repressors N-CoR and SMRT in the absence of ligand. Our current efforts are to explore GCNF function in the adult germ cells using tissue specific gene targeting to specifically knock out the GCNF gene in oocytes and spermatogenic cells. In addition, efforts are being made to identify the endogenous ligand that regulates GCNF's transcriptional properties.

A-ring reduced metabolites of 19-nor synthetic progestins as subtype selective agonists for ER alpha.

It has previously been demonstrated that 19-nor contraceptive progestins undergo in vivo and in vitro enzyme-mediated A-ring double bond hydrogenation. Bioconversion of 19-nor progestins to their corresponding tetrahydro derivatives results in the loss of progestational activity and acquisition of estrogenic activities and binding to the ER. Herein, we report subtype-selective differences in ligand binding and transcriptional potency of nonphenolic synthetic 19-nor derivatives between ER alpha and ER beta. In this study, we have examined both ER- and PR-mediated transcriptional activity of a number of A-ring chemically reduced derivatives of norethisterone and Gestodene. Double bond hydrogenation decreased the transcriptional potency of norethisterone and Gestodene through both PR isoforms with a 100- to 1,000-fold difference, respectively. In terms of the effects of norethisterone and Gestodene and their corresponding 5 alpha-dihydro (5 alpha-norethisterone and 5 alpha-Gestodene), or 3 alpha,5 alpha-tetrahydro or 3 beta,5 alpha-tetrahydro derivatives (3 alpha,5 alpha-norethisterone/3 alpha,5 alpha-Gestodene and 3 beta,5 alpha-norethisterone/3beta,5 alpha-Gestodene, respectively) on estrogen-mediated transcriptional regulation, the 3 beta,5 alpha-tetrahydro derivatives of both norethisterone and Gestodene showed the highest induction when HeLa cells were transiently transfected with an expression vector for ER alpha. This activity could be inhibited with tamoxifen. These compounds did not activate gene transcription via ER beta, and none of them showed antagonistic activities through either ER subtype. The 3 beta,5 alpha-tetrahydro derivatives of both norethisterone and Gestodene were active in other cells in addition to HeLa cells and activated reporter expression through the oxytocin promoter. In summary, two ER alpha selective agonists have been identified. These compounds, with ER alpha vs. ER beta selective agonist activity, may be useful in evaluating the distinct role of these receptors as well as in providing useful insights into ER action.

Physiological function of the orphans GCNF and COUP-TF.

Orphan nuclear receptors are members of the nuclear receptor superfamily of ligand-activated transcription factors for which ligands and functions have not been identified. Since the cloning of the original orphans, ligands have been identified for several orphan receptors that heterodimerize with the retinoid X receptor and are no longer classified as orphan receptors. Considering the central role that nuclear receptors play in differentiation, development, metabolic regulation, homeostasis and disease, it is crucial that we understand the roles of the remaining orphans. However, the identification of ligands for those orphans that form homodimers has proven more difficult. Thus, to gain greater insight into the functions of orphan receptors, gene targeting has been used to knock out these factors and study mouse development in their absence. Here we will review the progress made in understanding the roles of the orphans GCNF and the COUP-TFs with the use of gene knockouts.

Loss of orphan receptor germ cell nuclear factor function results in ectopic development of the tail bud and a novel posterior truncation.

The dynamic embryonic expression of germ cell nuclear factor (GCNF), an orphan nuclear receptor, suggests that it may play an important role during early development. To determine the physiological role of GCNF, we have generated a targeted mutation of the GCNF gene in mice. Germ line mutation of the GCNF gene proves that the orphan nuclear receptor is essential for embryonic survival and normal development. GCNF(-/-) embryos cannot survive beyond 10.5 days postcoitum (dpc), probably due to cardiovascular failure. Prior to death, GCNF(-/-) embryos suffer significant defects in posterior development. Unlike GCNF(+/+) embryos, GCNF(-/-) embryos do not turn and remain in a lordotic position, the majority of the neural tube remains open, and the hindgut fails to close. GCNF(-/-) embryos also suffer serious defects in trunk development, specifically in somitogenesis, which terminates by 8.75 dpc. The maximum number of somites in GCNF(-/-) embryos is 13 instead of 25 as in the GCNF(+/+) embryos. Interestingly, the tailbud of GCNF(-/-) embryos develops ectopically outside the yolk sac. Indeed, alterations in expression of multiple marker genes were identified in the posterior of GCNF(-/-) embryos, including the primitive streak, the node, and the presomitic mesoderm. These results suggest that GCNF is required for maintenance of somitogenesis and posterior development and is essential for embryonic survival. These results suggest that GCNF regulates a novel and critical developmental pathway involved in normal anteroposterior development.

The oestrogenic effects of gestodene, a potent contraceptive progestin, are mediated by its A-ring reduced metabolites.

Gestodene (17 alpha-ethynyl-13 beta-ethyl-17 beta-hydroxy-4, 15-gonadien-3-one) is the most potent synthetic progestin currently available and it is widely used as a fertility regulating agent in a number of contraceptive formulations because of its high effectiveness, safety and acceptability. The observation that contraceptive synthetic progestins exert hormone-like effects other than their progestational activities, prompted us to investigate whether gestodene (GSD) administration may induce oestrogenic effects, even though the GSD molecule does not interact with intracellular oestrogen receptors (ER). To assess whether GSD may exert oestrogenic effects through some of its neutral metabolites, a series of experimental studies were undertaken using GSD and three of its A-ring reduced metabolites. Receptor binding studies by displacement analysis confirmed that indeed GSD does not bind to the ER, whereas its 3 beta,5 alpha-tetrahydro reduced derivative (3 beta GSD) interacts with a relative high affinity with the ER. The 3 alpha,5 alpha GSD isomer (3 alpha GSD) also binds to the ER, though to a lesser extent. The ability of the A-ring reduced GSD derivatives to induce oestrogenic actions was evaluated by the use of two different molecular bioassays: (a) transactivation of a yeast system co-transfected with the human ER alpha (hER alpha) gene and oestrogen responsive elements fused to the beta-galactosidase reporter vector and (b) transactivation of the hER alpha-mediated transcription of the chloramphenicol acetyl transferase (CAT) reporter gene in a HeLa cells expression system. The oestrogenic potency of 3 beta GSD was also assessed by its capability to induce oestrogen-dependent progestin receptors (PR) in the anterior pituitary of castrated female rats. The results demonstrated that 3 beta GSD and 3 alpha GSD were able to activate, in a dose-dependent manner, the hER alpha-mediated transcription of both the beta-galactosidase and the CAT reporter genes in the yeast and HeLa cells expression systems respectively. In both assays the 3 beta derivative of GSD exhibited a significantly greater oestrogenic effect than its 3 alpha isomer, while unchanged GSD and 5 alpha GSD were completely ineffective. Neither 3 beta GSD nor 3 alpha GSD exhibited oestrogen synergistic actions. Interestingly, the pure steroidal anti-oestrogen ICI-182,780 diminished the transactivation induced by 3 beta GSD and 3 alpha GSD in the yeast expression system. Furthermore, administration of 3 beta GSD resulted in a significant increase of oestrogen-dependent PR in the anterior pituitaries of castrated rats in comparison with vehicle-treated animals. The characteristics of the 3 beta GSD-induced PR were identical to those induced by oestradio benzoate. The overall results demonstrate that 3 beta GSD and its 3 alpha isomeric alcohol specifically bind to the ER and possess a weak intrinsic oestrogenic activity, whereas unmodified GSD does not. The data contribute to a better understanding of the GSD mechanism of action and allow the hypothesis to be advanced that the slight oestrogenlike effects attributable to GSD are mediated by its non-phenolic, tetrahydro reduced metabolites.

Mouse protamine genes are candidate targets for the novel orphan nuclear receptor, germ cell nuclear factor.

Proper expression of the protamine genes is an important event in the terminal differentiation of the male gametes in mammals. Here we present evidence that the novel orphan member of the nuclear receptor gene superfamily, Germ Cell Nuclear Factor (GCNF), may play a role in the regulation of these genes. Previously, we have shown that high-level expression of GCNF mRNA is restricted to spermatids (stages 1-8) in the adult male mouse, which makes it temporally and spatially available to regulate the mouse protamine genes. Furthermore, we have previously identified a sequence to which GCNF can bind, which consists of a direct repeat of the core halfsite AGGTCA with zero base pairs spacing the repeats (DRO). We have identified several genes that contain DRO sequences in their 5' promoter regions, including the protamines. The mouse protamine 1 and 2 (Prm1 and Prm2) genes therefore are potential target genes for GCNF regulation. We show that GCNF binds to one of the two DRO sequences in the Prm1 promoter, and to the DRO sequence in the Prm2 promoter in a specific manner. Furthermore, by using antibodies directed against GCNF, we detect endogenous GCNF in testis nuclear extracts and elutriated round spermatid nuclear extracts in Western blots. Additionally, by using these antibodies in gel-shift assays, we show that this endogenous GCNF can bind to both the Prm1 and Prm2 promoters. This evidence supports the hypothesis that GCNF mediates a novel signaling pathway, two targets of which may be the Prm1 and Prm2 genes in spermatids.

Germ cell nuclear factor is a response element-specific repressor of transcription.

We have shown that the orphan receptor Germ Cell Nuclear Factor (GCNF) binds to a direct repeat of the sequence AGGTCA with zero base pair spacing (DR0). Here, we further characterize the binding characteristics of GCNF. We demonstrate that GCNF binds specifically to DR0s as a homodimer, and does not bind with high affinity to DR1-DR6 sequences. GCNF is the first nuclear receptor shown to bind specifically to DR0s. The wild type GCNF is unable to transactivate the reporter plasmid DR0(2)tkCAT. Lacking a ligand to activate GCNF, we fused the activation domain from the viral protein VP16 to GCNF, and observed activation of DR0(2)tkCAT. This activation is specifc to DR0s, and is not observed when that sequence is replaced by DR1-DR6 sequences. In addition GCNF does not transactivate through an SF-1 response element. At increasing concentrations, wild type GCNF is able to repress basal transcription. Repression is again specific to DR0s. The preference of GCNF for the DR0 sequence both in vitro and in transfections suggests that GCNF defines a novel nuclear receptor signaling pathway.

Cloning, expression analysis and chromosomal localization of the human nuclear receptor gene GCNF.

Germ cell nuclear factor (GCNF) is an orphan member of the nuclear receptor gene superfamily. We report the cloning of a cDNA encoding a new variant of human GCNF from human testis and its expression analysis. Southern blot analysis of the human genomic DNA indicates that the GCNF gene is not closely related to other members within the nuclear receptor superfamily. Chromosomal localization of the GCNF gene shows that the gene is located on chromosome 9 at the locus q33-34.1. In situ hybridization analysis of GCNF expression in the testis shows that human GCNF is expressed exclusively in germ cells.

Characterization of germ cell-specific expression of the orphan nuclear receptor, germ cell nuclear factor.

Nuclear receptors, such as those for androgens, estrogens, and progesterones, control many reproductive processes. Proteins with structures similar to these receptors, but for which ligands have not yet been identified, have been termed orphan nuclear receptors. One of these orphans, germ cell nuclear factor (GCNF), has been shown to be germ cell specific in the adult and, therefore, may also participate in the regulation of reproductive functions. In this paper, we examine more closely the expression patterns of GCNF in germ cells to begin to define spatio-temporal domains of its activity. In situ hybridization showed that GCNF messenger RNA (mRNA) is lacking in the testis of hypogonadal mutant mice, which lack developed spermatids, but is present in the wild-type testis. Thus, GCNF is, indeed, germ cell specific in the adult male. Quantitation of the specific in situ hybridization signal in wild-type testis reveals that GCNF mRNA is most abundant in stage VII round spermatids. Similarly, Northern analysis and specific in situ hybridization show that GCNF expression first occurs in testis of 20-day-old mice, when round spermatids first emerge. Therefore, in the male, GCNF expression occurs postmeiotically and may participate in the morphological changes of the maturing spermatids. In contrast, female expression of GCNF is shown in growing oocytes that have not completed the first meiotic division. Thus, GCNF in the female is expressed before the completion of meiosis. Finally, the nature of the two different mRNAs that hybridize to the GCNF complementary DNA was studied. Although both messages contain the DNA binding domain, only the larger message is recognized by a probe from the extreme 3' untranslated region. In situ hybridization with these differential probes demonstrates that both messages are present in growing oocytes. In addition, the coding region and portions of the 3' untranslated region of the GCNF complementary DNA are conserved in the rat.

Molecular mechanisms of COUP-TF-mediated transcriptional repression: evidence for transrepression and active repression.

COUP-TF, an orphan member of the nuclear receptor superfamily, has been proposed to play a key role in regulating organogenesis, neurogenesis, and cellular differentiation during embryonic development. Since heterodimierization is a common theme within the nuclear receptor superfamily and has been demonstrated to modulate transcriptional properties of heterodimeric partners via allosteric interactions, we have devised a strategy to examine the silencing function of COUP-TF in a heterodimeric context. We find that the intrinsic active repression function of COUP-TF is not affected by heterodimerization. Moreover, COUP-TF can transrepress the ligand-dependent activation of its heterodimeric partners without its own DNA binding site. Using receptor deletion mutants in transfection assays, we show that the region necessary for COUP-TF silencing function is not sufficient for its transrepression activity. Furthermore, our studies indicate that in addition to its active repression function, COUP-TF can repress several different types of activator-dependent transactivation. However, this active repression function of COUP-TF may be differentially regulated by some other activator(s). These studies provide new insights into the molecular mechanism(s) of COUP-TF-mediated repression.

The mouse bone morphogenetic protein-4 gene. Analysis of promoter utilization in fetal rat calvarial osteoblasts and regulation by COUP-TFI orphan receptor.

Bone morphogenetic protein-4 (BMP-4) is one of a member of related polypeptides that are important in bone formation and other developmental processes. We isolated the BMP-4 gene from a mouse genomic library and characterized the exon-intron structure and one of the candidate promoters. Two alternative 5'-noncoding exons, 1A and 1B, were identified by reverse transcription polymerase chain reaction assays. Quantitative competitive polymerase chain reaction using Exon 1A, Exon 1B, and Exon 3 primers indicate the 1A-containing transcript is the primary BMP-4 mRNA expressed in bone cell cultures. Primer extension analysis supports that 1A is the major promoter utilized in bone cell cultures as well as in 9.5-day mouse embryos. 1A promoter activity indicate selective DNA regions functional in bone cells. We found potential regulatory response regions in the 1A 5'-flanking region of the BMP-4 gene for the chicken ovalbumin upstream-transcription factor I (COUP-TFI). Specific binding to the COUP-TFI response regions in the BMP-4 1A promoter was demonstrated. By co-transfection of a COUP-TFI expression plasmid with the BMP-4 1A promoter in fetal rat calvarial osteoblasts, we demonstrated that COUP-TFI inhibits the BMP-4 promoter activity. This suggests that COUP-TFI could act as a silencer for BMP-4 transcription in vivo.

Cloning of a novel orphan receptor (GCNF) expressed during germ cell development.

We have cloned a novel member of the nuclear receptor superfamily that has been identified from complementary DNA libraries derived from mouse tissues using a low stringency cross hybridization strategy. The deduced protein sequence contains 495 amino acids and consists of the characteristic DNA-binding and ligand-binding domains of the nuclear receptor superfamily. The primary sequence of this new orphan is distinct from those of previously cloned members and subgroups. Analysis of the DNA-binding properties of the in vitro synthesized protein revealed that this new orphan receptor binds to the sequence TCAAGGTCA that includes the steroidogenic factor-1 half-site and direct repeat with 0 bp spacing elements. Northern blot and ribonuclease protection assays showed that the receptor was predominantly expressed in the testis. Results from in situ hybridization experiments confirmed this observation and showed it to be located in the spermatogenic cells. High level expression was also detected in developing oocytes in the ovary. Thus, high level expression of this gene is restricted to developing germ cells, the oocytes and spermatogenic cells. We speculate that this orphan receptor may be a molecule involved in regulating some aspect of meiosis, and that the major function of this factor is likely to be involved in the regulation of gene expression in germ cell development during gametogenesis. It has been designated germ cell nuclear factor.

A rapid one-step method to purify baculovirus-expressed human estrogen receptor to be used in the analysis of the oxytocin promoter.

We have produced a truncated form of the human estrogen receptor (hER) as a fusion protein with glutathione S-transferase (GST) in Spodoptera frugiperda (Sf) cells using the baculovirus expression vector (BEV) system. The protein is correctly produced and can be purified from crude whole-cell extracts by a single-step, batch-wise affinity-purification procedure. We show that this GST-hER fusion protein binds at its DNA-binding site specifically and in a hormone-inducible manner. Furthermore, we used the purified hER to analyze the complex estrogen response element (ERE) in the promoter of the oxytocin-encoding gene.

Negative regulation by the R2 element of the MHC class I enhancer in adenovirus-12 transformed cells correlates with high levels of COUP-TF binding.

The transcriptional down-regulation of the major histocompatibility complex (MHC) class I antigens in adenovirus type 12 (Ad12) transformed cells gives them the potential to escape immunosurveillance and to form tumors. The enhancer of the class I promoter is the target of transcriptional repression which is mediated by the E1A gene of Ad12. The R2 region within the class I enhancer acts as a negative element in Ad12-transformed cells and exhibits a stronger binding activity than is observed in nontumorigenic Ad5-transformed cells, which are not reduced in class I expression. The R2 element contains a nuclear hormone receptor half-site consensus sequence, AGGTCA, which is required for both the binding activity and the ability of R2 to act as a negative element in Ad12-transformed cells. In this study, we show that an orphan hormone receptor protein, COUP-TF, contributes to the differential R2 binding activity observed between Ad12- and Ad5-transformed cells. Additionally, COUP-TF was shown to bind as a dimer to the R2 element and to use the consensus AGGTCA as one half-site and its 3' flanking sequence as a probable second degenerate half-site. Since COUP-TF can act as a transcriptional repressor, we suggest that the higher COUP-TF binding activity to the R2 element in Ad12-transformed cells contributes to down-regulation of class I transcription and, consequently, tumorigenesis.

Detection of potential ligands for nuclear receptors in cellular extracts.

In response to external stimuli, steroid receptors are directly influenced to transactivate gene expression. Assuming they exist, identification of ligands for orphan steroid receptors is a key to understanding their physiology. In the orphan subgroup of the steroid receptor superfamily, the putative carboxyl terminal ligand-binding domain (LBD) is well conserved among members of the superfamily, which suggests a role in ligand binding. A consequence of ligand binding is the induction of a significant conformational change within the LBD which is necessary for the transactivation function. This characteristic conformational change can be detected by partial proteolytic digestion and has been localized by mutational analysis and epitopic mapping of the LBD using monoclonal antibodies. Based on this finding, a sensitive in vitro assay was developed for the rapid screening and identification of potential ligands for orphan receptors. We examined the patterns of conformational changes in the androgen receptor, glucocorticoid receptor, and progesterone receptor induced by binding of their cognate agonists and antagonists. We demonstrated that the conformational changes induced by ligands can serve as characteristic and reliable markers to distinguish between the ligand-bound and apoprotein states of a receptor. The sensitivity and feasibility of employing this assay to detect new endogenous ligands using fractionated cellular extracts were also tested. The results strongly suggest that unknown compounds can be defined as potential ligands for orphan receptors using this approach.

Repression of estrogen-dependent stimulation of the oxytocin gene by chicken ovalbumin upstream promoter transcription factor I.

The orphan receptor chicken ovalbumin upstream promoter transcription factor I (COUP-TF I) fully prevented not only the activation of the oxytocin gene by retinoic acid and thyroid hormone but also completely repressed the estrogen-dependent stimulation in transfected P19 EC cells. DNase I footprinting showed that the COUP-TF I protein bound to the 5'-flanking region of the oxytocin gene at the site of the distal composite hormone response element, which mediates the responses to estrogen, retinoic acid, and thyroid hormone. Electrophoretic mobility shift assay using this composite hormone response element as probe showed that COUP-TF I and the estrogen receptor competed for binding but did not form a heterodimer. The binding by COUP-TF I was stronger than the binding of the estrogen receptor. Thus, the mechanism of repression involves occupancy of integrated binding sites. By mutagenesis of the composite hormone response element, the COUP-TF I binding site and the estrogen response element could be separated, resulting in functional dissociation of the repressive action of COUP-TF I and the induction by estrogen. The results show that repression of gene expression by COUP-TF I is not limited to receptors that act through heterodimerization but also extends to the homodimer-forming estrogen receptor in a context-dependent manner. This interaction between COUP-TF I and the estrogen receptor may provide a physiological mechanism of selective antagonism of gene regulation by estrogens.